The presence of a foreign antigenic substance in the body typically gives rise to an immune response. Antibodies specific for the substance are secreted into the blood stream. Diagnostic tests revealing exposure of an animal to an infectious agent rely upon in vitro detection of antibodies specific for antigens expressed by such an infectious agent. In a typical immunoassay, antibodies contained in a serum sample are brought into contact with an antigen preparation. The resulting immune complex is detected in a variety of assay formats including enzyme-linked immunosorbant assay (ELISA), agglutination tests, complement fixation assay, etc.
Many of the assay formats involve adsorption of antigens into a solid phase of such as a plastic bead or particle, a plastic well, or the surface of a plastic or glass tube. The serum containing antigen-specific antibodies are contacted within antigen coated onto solid phase, and washed to remove unreacted serum components. The solid phase-bound antibodies are then detected, for example, by reacting with a species-specific antibody bearing a reporter molecule, washing to remove unreacted reagent, and finally adding a reagent to activate the reporter.
It will be apparent that such assays involve complex binding relationships. There is a tendency for nonspecific antibody to stick to the solid phase, giving rise to false positive tests. Even the best ELISA assays will produce a low level of false positive results. For some of the agglutination assays, specificities of less than 70 percent are obtained.
There have been attempts to eliminate the solid phase artifacts referred to above by detection of analyte antibodies entirely in liquid phase. Termed homogeneous assays, these systems rely upon measuring some changed physico-chemical parameter brought about by molecular interactions. For a review of homogeneous assays systems, see Jenkins, S. H., J. Immunol. Meth., 150: 90 (1992). For applications to macromolecules, see Hosoda, et al., J. Immunol. Meth., 121: 121 (1989). For review of the theory and practice of fluorescence polarization, a particularly elegant homogeneous system, see M. E. Jolley, J. Anal. Toxicol., 5: 236 (1981).
In the field of infectious disease diagnostics, there is a strong need for assays which have extremely high specificities. In particular, in veterinary diagnostics there are the so-called xe2x80x9cprogramxe2x80x9d diseases, which official agencies such as the United States Dept. of Agriculture or Agriculture and Agri-Food Canada administer, and whose eradication is part of their official mandate. The regulations require that a very large number of animals be tested to ensure that new infection is promptly detected and eliminated, and to prove absence of infection.
One of the most important program diseases of livestock is bovine brucellosis. This disease is caused by Brucella abortus, a Gram negative bacterium inhabiting the genital tract of cattle. Infected heifers are highly susceptible to abortion, and the economic loss to the cattle industry has been prodigious. Through concerted international efforts to screen for infected animals, and disposal of test positive animals at slaughter, the disease is essentially eradicated in Canada, and the incidence in the U.S. has diminished to less than 150 infected herds. However, Mexico is a persistent reservoir of infection, and movement of cattle in commerce to the U.S. poses a continual risk of new disease. Also, wild animals such as deer and elk are a reservoir of potential new disease in domestic livestock.
As more states become brucellosis-free, and as the overall incidence of disease continues to decline there will be a new emphasis on surveillance programs. In a surveillance situation sensitivity of the assay becomes less important than specificity (so long as the test is sufficiently sensitive to detect a typical reactor). In surveillance testing, false positive tests are troublesome, since any positive test cannot be ignored. For any such positive test, it is official procedure to trace the animal, retest it, and subject it to quarantine. This is extremely costly, and a waste of limited resources.
A large number of cattle in the United States are vaccinated for brucellosis in calfhood, with about 73 percent gaining protective immunity from superinfection with field strains of B. abortus. Unfortunately, none of the currently available tests distinguish with high accuracy between true field strain infected animals and vaccinated ones. Specificity is as low as about 50 percent for agglutination tests and up to as high as 97 percent for a C-ELISA assay. In a surveillance program, 3 percent false positives for calfhood vaccinates is a large number in view of the several million cattle tested annually in the U.S. In fact, the high incidence of positive tests for vaccinates, has led some states to repeal their mandatory calfhood vaccination laws, thus depriving the industry of one otherwise valuable tool in the eradication of the disease.
Conventional assays for detection of brucellosis include the ubiquitous agglutination card test in the U.S., the BPAT test in Canada, various versions of a complement fixation test, the Rivanol test, milk ring test (for dairy specimens), and several forms of ELISA including a highly sensitive and specific competitive ELISA developed by Agriculture and Agri-Food Canada. A solid phase bead-based test known as PCFIA is a high throughput automated test used in several official laboratories in the U.S. Many of these tests are commonly used together on the same samples because no one of the conventional, generally available tests detect all the antibody isotypes typically found in field specimens. For a general review of conventional brucellosis tests, see Nielsen, K. and Duncan, J. R., eds., Animal Brucellosis, CRC Press: 1990, Chapt. 8.
Brucellosis is but one example of veterinary disease for which a need exists for a sensitive yet highly specific assay approaching 100 percent. Other diseases include tuberculosis (M. bovis), bovine leukosis, leptospirosis, foot and mouth disease (Africa and South America), and salmonellosis.
In the present invention, a fluorophore-conjugated antigen dissolved freely in solution reacts with antibodies contained in a diluted serum sample to form a immune complex. The immune complex is detected by a change in fluorescence polarization.
It is an object of the invention to provide a homogeneous assay for detecting antibody to selected antigens, thereby eliminating complex, time-consuming adsorption, washing, and detection steps. The assay involves only a single reagent, and a single step reaction in which an immune complex is detectable by a simple fluorescence polarization measurement.
It is a further object of the invention to provide an assay which has extremely high specificity, with less than 0.1 percent down to 0 percent false positive tests, while maintaining a sensitivity of greater than 98 percent.
It is a still further object to provide an assay in which the antigen reagent can be prepared in large reproducible lots of highly uniform composition and performance.
The antigen reagent is also very stable with virtually indefinite lifetime when stored at refrigerated temperatures in the presence of a microbial inhibitor such as sodium azide.
The present method of the invention detects antibodies to bacterial antigens present in fluid comprising combining fluid which may contain antibodies to a bacterial antigen with a fluorophore-conjugated oligosaccharide antigen derived from the lipopolysaccharide antigen derived from the lipopolysaccharide fraction of a bacterial cell wall to form a mixture, incubating the mixture for a time sufficient to form an immune complex, and measuring the extent of formation of the immune complex by comparing the fluorescence polarization after complex formation to an unreacted control value.
The invention provides fluorophore-conjugated antigens comprising oligosaccharides derived from the lipopolysaccharide fraction of a bacterial cell wall. The fluorophore-conjugated oligosaccharide is one embodiment derived from the O-antigen chain of the cell wall lipopolysaccharide fraction from bacteria, such as Gram negative bacteria, having us O-antigen chain.
The invention similarly comprises a homogeneous immunoassay in which antibodies to an antigen are reacted with an antigen to form an immune complex, the improvement to previous technology being a fluorophore-conjugated oligosaccharide antigen of bacterial cell wall forming a fluorescence polarization detectable immune complex with antibodies specific thereto.
The fluorophore-conjugated oligosaccharide antigens may be derived from any bacterium having a lipopolysaccharide cell wall fraction, as antigens derived from Gram negative bacteria such as B. abortus, Leptospira spp, or Salmonella spp. The fluorophore will have an emission life of 1 to 10 nanoseconds, and its emission may vary over a wave length of 400 to 800 nanometers. In the present invention, the fluorophore fluorescein is efficacious.